There is growing evidence that stem cells age, and that their doing so may contribute to age-related alteration in tissue maintenance and repair. However, age-related changes in stem cells may differ as a function of cellular turnover in the tissue in which they reside. The tractability of the testis as a model system makes it possible to address the effect of age on stem cell function in a slowly-renewing tissue. Leydig cells, the testosterone-producing cells of the testis, turn over very infrequently. The presence of SLCs in the testes of both neonatal and adult rats, and our ability to isolate and culture these cells, make it possible to determine whether there are age-related changes in SLC function. Additionally, our ability to transplant these cells into Leydig cell-depleted testes makes it possible to determine whether the external environment of the SLCs affects their function. In this application, we propose experiments, both in vitro and in vivo, to determine whether aging results from intrinsic changes in the cells or from the impairment of SLC function by age-related changes in their niche. Two specific aims are proposed, to be carried out over a period of four years. First, we will conduct array analyses of isolated SLCs from the testes of young adult, aged and neonatal testes, and additionally will examine the relative abilities of isolated SLCs to self-renew or to differentiate in vitro. In the second aim, in vivo studies will be carried out to determine whether age and the Leydig cell niche affect SLC proliferation and differentiation. For these studies, SLCs will be genetically tagged with green fluorescent protein so that the cells can be followed after their transplantation. SLCs from the testes of early postnatal, young adult or aged rats will be transplanted into the Leydig cell-depleted testes of adult rats, or SLCs from neonatal testes into the Leydig cell-depleted testes of young adult versus aged rats. The rates at which the transplanted cells proliferate and the numbers of proliferating cells will be measured. The results, taken together, will indicate whether the SLCs carry out a program that is inherent to the cells or is influenced by the environment of the niche. If we find that the function of the SLCs are influenced little by age or by the external environment, we will suggest that if adult stem cells are to be of utility for therapeutic purposes, the cells of choice should come from tissues that turn over little.